1. Field of the Invention
The present invention generally relates to ricin toxin. In particular, the present invention relates to ricin vaccines as well as methods of making and using thereof.
2. Description of the Related Art
Ricin is a very toxic protein obtained from the castor bean, Ricinus communis, Euphorbiaceae. Ricin is a heterodimer comprising an A chain and a B chain joined by a disulfide bond. Ricin A chain (RTA) is an N-glycosidase enzyme that irreversibly damages a specific adenine base from 28S rRNA. Once the rRNA has been damaged, the cell cannot make protein and will inevitably die (cytotoxicity). As RTA exhibits this type of destructive catalytic activity, RTA is commonly referred to as a type II ribosome inactivating protein (RIP). See Lord, et al. (1991) Semin. Cell Biol. 2(1):15-22. RTA has been coupled with a targeting moiety to selectively destroy target cells such as tumor cells. See U.S. Pat. Nos. 4,80,457; 4,962,188; and 4,689,401; see also Vitetta et al. (1993) Trends Pharmacol. Sci. 14:148-154 and Ghetie & Vitetta (1994) Cancer Drug Delivery 2:191-198.
The toxic consequences of ricin are due to the biological activity of RTA. Ricin B chain (RTB) binds the toxin to cell surface receptors and then RTA is transferred inside the cell where inhibition of ribosome activity occurs. The human lethal dose of ricin toxin is about 1 μg/kg. As highly purified ricin is commercially available, the use of ricin toxin in biological warfare and terrorism is highly possible and probable. Unfortunately, there is no effective antidote for toxic exposure to ricin. Thus, attempts have been made to provide vaccines against ricin intoxication.
Ricin vaccines have been prepared by isolating the natural toxin from castor beans, and treating the toxin with harsh chemicals, such as typically formaldehyde, to reduce the toxic activity. See Hewetson, et al. (1993) Vaccine 11(7):743-746; Griffiths, et al. (1995) Hum. Exp. Toxicol. 14(2):155-164; Griffiths, et al. (1999) Vaccine 17(20-21):2562-2568; and Yan, et al. (1996) Vaccine 14(11):1031-1038. These first generation vaccines are called “toxoid” vaccines as they are made directly from natural toxin itself. The current toxoid vaccine suffers from several important limitations that include: (1) the presence of both RTA and RTB; (2) the presence of trace amounts of active RTA and RTB; (3) the possibility that denatured toxoid could refold and, thereby, revert to the active form; (4) side-effects that arise from the presence of the harsh chemicals used in the vaccine formulation; (5) heterogeneity in the final vaccine product arising from the process of formaldehyde treatment; and (6) heterogeneity in the final vaccine resulting from the natural structural variations of ricin protein found in castor beans. See Despeyroux, et al. (2000) Anal. Biochem. 279(1):23-36.
The second generation ricin vaccines comprise wild-type (wt) RTA, but not RTB. See U.S. Pat. No. 5,453,271. Early efforts centered on isolating the whole toxin from castor beans, and then purifying RTA from RTB. Unfortunately, there are major problems associated with the use of natural or wt RTA as a vaccine such as: (1) heterogeneity at the level of protein composition (Despeyroux, et al. (2000) supra); (2) heterogeneity at the level of sugar composition (N-linked and/or o-linked carbohydrates covalently attached to the polypeptide backbone) (Despeyroux, et al. (2000) supra); (3) retention of naturally toxic N-glycosidase-rRNA enzymatic activity; (4) the poor solubility of isolated RTA in the absence of RTB, as evidenced by protein aggregation under physiological conditions; (5) rapid clearance circulation by the liver, thereby reducing the effectiveness of the vaccine (Wawrzynczak, et al. (1991) Int. J. Cancer 47:130-135); and (6) causing lesions of the liver and spleen.
Heterogeneity (variability) in a vaccine is undesirable as standardized and reproducible vaccine lots are necessary for regulatory compliance and approval. To reduce the heterogeneity of RTA vaccines, deglycosylated RTA (dgRTA) vaccines were produced. See International patent publication WO 00/53215. Unfortunately, dgRTA is still poorly soluble. Additionally, both dgRTA and wt recombinant RTA (rRTA) retain toxic N-glycosidase-rRNA enzymatic activity, which poses a safety risk. See Blakey, et al. (1987) Cancer Res. 47(4):947-952; Foxwell, et al. (1987) Biochim. Biophys. Acta. 923(1):59-65; Soler-Rodriguez, et al. (1992) Int. J. Immunopharmacol. 14(2):281-291; and Schindler, et al. (2001) Clin. Cancer Res. 7(2):255-258. Attempts at eliminating the toxic enzymatic activity of wt RTA gave rise to the third generation of ricin vaccines.
The third generation ricin vaccines are based on the active site of RTA that comprises the amino acid residues that interact directly with, or are within about five angstroms from, the bound ribosomal RNA substrate. Specifically, these mutant substitution RTA vaccines are based on recombinant DNA technology and substitute amino acids of wt RTA in order to reduce N-glycosidase-rRNA activity. See Ready, et al. (1991) Proteins 10(3):270-278; Kim, et al. (1992) Biochem. 31:3294-3296; Roberts, et al. (1992) Targeted Diag. Ther. 7:81-97; Frankel, et al. (1989) Mol. Cell. Biol. 9(2):415-420; and Gould, et al. (1991) Mol. Gen. Genet. 230(1-2):81-90.
Unfortunately, these mutant substitution RTA vaccines are problematic because unwanted changes often occur in the protein structure and render the protein unstable. Self-organization of the native RTA tertiary fold is optimized by the electrostatic charge balance of the active site cavity. See Olson (2001) Biophys. Chem. 91(3) 219-229. Thus, amino acid substitutions that alter the charge balance lead to structural reorganization coupled with a reduction in protein-fold stability. For example, disrupting the ion-pair between amino acid residues, Glu-177 and Arg-180, at the active site cavity by replacing the arginine with a histidine affects the global stability of the protein if the imidazole ring is deprotonated. See Day, et al. (1996) Biochem. 35(34): 11098-11103. The more stable form of mutant substitution RTA R180H, reduces the overall enzymatic activity about 500-fold, yet remains cytotoxic.
Another example of a failed substitution was mutant substitution RTA E177A. The x-ray crystallographic structure of RTA E177A demonstrates a remarkable rescue of electrostatic balance in the active site, achieved by the rotation of a proximal glutamic acid into the vacated space. See Kim, et al. (1992) supra. Despite the non-conservative substitution, the free energy of denaturation for RTA E177A was anticipated from modeling studies to be two-fold more favorable than the conservative replacement of mutant substitution RTA E177Q See Olson (2001) supra. In terms of expression levels, RTA E177Q is far less well behaved than wt RTA. See Ready, et al. (1991) supra. Additionally, both RTA E177A and RTA E177Q remain active enzymes, thereby indicating plasticity in obtaining the catalytic transition-state. See Schlossman, et al. (1989) Mol. Cell. Biol. 9(11):5012-5021; and Ready, et al. (1991) supra.
Thus, a need still exists for a vaccine that is stable, safe and effective against ricin intoxication.